Leydig cell receptors for luteinizing hormone releasing hormone and its agonists and their modulation by administration or deprivation of the releasing hormone

Leydig cells isolated from adult rat testes bound ¹²⁵I-labelled luteinizing hormone releasing hormone (LHRH) agonist with high affinity (K_A=1.2 × 10⁹M) and specificity. LHRH and the 3–9 and 4–9 fragments of LHRH agonist competed for binding sites with ¹²⁵I-LHRH agonist but with reduced affinities, whereas fragments of LHRH, and oxytocin and TRH were largely inactive. Somatostatin inhibited binding at high (10^?4M) concentrations but was inactive at 10^?6M and less. Pretreatment of rats for 7 days with 5 μg/day of LHRH agonist reduced binding of ¹²⁵I-LHRH agonist to Leydig cells $\text{in vitro}$ by 25%, whilst inhibition of endogenous LHRH by antibodies for 7 days caused a 40% decrease.     

Human placental receptors for luteinizing hormone releasing hormone

The GTPase activity of the tubulin-colchicine complex has been studied at different tubulin-colchicine concentrations. The specific activity was found to decrease at low concentrations. Several hypothesis accounting for this observation have been discarded, and the activation via collisions between two molecules of tubulin has been considered as a possible model explaining the origin and observed concentration dependence of the GTPase activity. The activation of tubulin-colchicine by unliganded tubulin or tubulin-podophyllotoxin has been investigated within this model which emphasizes the connection between some specific tubulin-tubulin interactions and the conformation of the exchangeable nucleotide site on tubulin.     

Decreased in vivo protein and phospholipid methylation after in vivo elevation of brain S-adenosyl-homocysteine

The administration of adenosine together with homocysteine resulted in a dose-related elevation of cerebral S-adenosyl-L-homocysteine without concomitant perturbation of S-adenosyl-L-methionine levels. The adenosine + homocysteine treatment also decreased the incorporation of labile and stable methyl groups into brain proteins. Brain [³H]-phosphatidyl N,N-dimethylethanolamine and [³H]-phosphatidylcholine were also significantly decreased while [³H]-phosphatidyl-N-monomethylethanolamine remained unchanged. The data indicate that elevated brain S-adenosylhomocysteine can markedly and selectively inhibit the $\text{in vivo}$ methylation of brain proteins and phospholipids.     

Sizing of two bacteriophage T4 specific messenger ribonucleic acids formed in vitro and in vivo

Messenger RNA for two T4 specific enzymes, deoxynucleotide kinase and α glucosyltransferase, have been sized by sedimentation on sucrose density gradients. The sedimentation constants of transferase and kinase mRNAs formed $\text{in vitro}$ were 21.5S and 14.5S respectively, regardless of the duration of incubation up to 20 min. Although the kinase mRNA isolated from cells infected with T4 phage for 10 min was the same size as found $\text{in vitro}$ (14.5S), the transferase mRNA was found in a segment approximating the size of the kinase mRNA (14.5S). The experiments indicate that α glucosyltransferase mRNA is formed first as a polycistronic message and is then processed to the smaller unit.     

Monitoring the stereospecificity of morphine action in vivo and in vitro through brain membrane lipid fluidity

Differential scanning calorimetry of crude brain mitochondrial lipids obtained from control and morphine treated rats was carried out and the lipid phase transition measured. Morphine treatment resulted in a significant decrease in the temperature range and enthalpy of the phase transition. This effect was found to be dose dependent and reversible both $\text{in vivo}$ and $\text{in vitro}$ by naloxone. Studies with levorphanol and dextrorphan demonstrated stereospecificity. Furthermore, the ether precipitable fraction of total lipid extracts was shown to mediate the opiate response.     

Extremely rapid degradation of [3H] methionine-enkephalin by various rat tissues in vivo and in vitro

Thirty sec after the intrajugular injection of [³H] methionine-enkephalin (met-enkephalin) in the rat, the radioactivity was already distributed in an apparent volume of 53 ml and the metabolic clearance rate calculated from the characteristics of the plasma disappearance curve was 10 ml/min. As shown by partition chromatography plasma extracts obtained 15 sec after injection of [³H] met-enkephalin, only 5% of the total radioactivity migrated as the intact pentapeptide, while no detectable intact pentapeptide remained 2 min after injection, thus indicating a half-life of [³H] met-enkephalin of the order of 2 to 4 sec. Incubation of rat cerebral tissue with [³H] met-enkephalin indicates that the first step in the breakdown of met-enkephalin in both plasma and brain tissue is cleavage of the Tyr-Gly amide bond. These data offer an explanation for the low activity of met-enkephalin after intraventricular or intravenous administration.     

Discordant regulation by luteinizing hormone of ornithine decarboxylase activity and testosterone production in isolated rat testicular cells invitro

Possible functional relationship between luteinizing hormone-stimulated ornithine decarboxylase and testosterone production was examined in rat testicular interstitial cells $\text{in}\text{vitro}$. Although luteinizing hormone enhanced both ornithine decarboxylase activity and testosterone production at a similar physiological dose range, we found dissociation in the two responses in terms of their temporal aspect and the way they were affected by an irreversible inhibitor of ornithine decarboxylase, alpha-difluoromethylornithine, and protein synthesis inhibitor cycloheximide. The results suggest that there appears to be no causal coupling between luteinizing hormone-stimulated enzyme activity and testicular steroidogenesis.     

Effect of metoclopramide on rat prolactin secretion in vivo

Metoclopramide, a potent antagonist to apomorphine, was used to the rats $\text{in vivo}$ to determine its effect on the release of prolactin. A single i.p. injection of metoclopramide at 10 or 100 μg/100 g b.w. under urethane anesthesia increased serum prolactin levels by 1.6 or 7.2 fold, respectively, compared with basa levels. This prolactin increase was completely abolished by 2-bromo-α-ergocryptine (CB-154).These data suggest that metoclopramide stimulates prolactin secretion in rat and this secretion is abolished by dopaminergic stimulant.     

Regulation of argF mRNA synthesis,performed in vitro

The specific synthesis of $\text{arg}$F mRNA directed by the $\text{arg}$F gene carried on the specialized transducing bacteriophage $\text{λh80C}{}_1\text{857darg}$F, performed $\text{in vitro}$, is described with the use of an S180 extract from a strain carrying $\text{arg}$R^?. Synthesis of $\text{arg}$F mRNA is biphasic at approximately 7 minutes. The regulation of $\text{arg}$F mRNA synthesis by the specific arginine holorepressor present in an S180 extract prepared from a strain carrying the $\text{arg}$R⁺ allele is described.     

The in vivo inhibition of gaba-transaminase by gabaculine

The naturally occurring amino acid gabaculine ((?)-5-amino-1,3 cyclohexadiene carboxylic acid) is a potent irreversible inhibitor of mouse brain γ-aminobutyric acid (GABA)-α-ketoglutaric acid transaminase. When administered I.P. gabaculine, irreversibly inhibits the mouse brain enzyme in a time dependent fashion. Concomitant with this inhibition is a rise in endogenous brain GABA levels. Administration of gabaculine at a concentration of 100 mg/kg mouse leads to the complete inhibition of the enzyme after 4 hrs. Brain levels of GABA continually rise after the administration of the drug. After 20 hrs they are 15–20 times higher than levels in the untreated animals.     

Inhibition of natural killer cell activity in vitro by alcohols

The effect of the addition of small quantities of alcohols to cocultures of natural killer effector cells from $\text{C57BL}\text{6}$ mice and BDIX rats with YAC-1 tumor cell targets has been studied. The order of inhibition of NK cell-mediated killing is 1-butanol > 1-propanol > 2-propanol > ethanol > methanol. The inhibition of killing due to the addition of alcohol correlates with decreases of effector-target cell binding. Therefore caution should be exercised in interpreting results of cellular experiments in which these alcohols have been used to solubilize inhibitors.     

Effect of ovarian hormone pretreatment on gallbladder motility in vitro

Adult male guinea pigs were pretreated with estrogen, progesterone, or estrogen plus progesterone and the $\text{in vitro}$ contractile response of the gallbladder to cholinergic stimulation examined. The data were compared with results obtained from control animals. Progesterone pretreatment was associated with a significant decrease in the maximal contractile response of the tissues and with a significant increase in the dose of acetylcholine needed to produce a threshold response. Estrogen pretreatment significantly decreased the threshold dose requirements but had no effect on maximum tension development. In addition, estrogen pretreatment antagonized the inhibitory effect of progesterone pretreatment. The data support the hypothesis that the ovarian steroid hormones can affect gastrointestinal smooth muscle. Furthermore, the hormones appear to exert independent and opposite effects on gallbladder motility. Additional studies will be required to determine the physiological significance of these observations.     

Gonadotropin releasing hormone (GnRH) is not degraded by intact pituitary tissue in vitro

The possible degradation of GnRH by intact pituitary tissue was investigated. Trypsin-, collagenase- and mechanically dispersed pituitary cells in culture and fresh pituitaries cut into eight segments were incubated with specifically tritiated GnRH or unlabeled GnRH which were detected by HPLC and RIA in incubation media. Degradation of GnRH could not be detected by either method during incubations with any of the pituitary cell cultures or fresh tissue. The results suggest that pituitary degradation is not involved in the regulation of circulating levels of GnRH.     

Inactivation of aromatase in vitro by 4-hydroxy-4-androstene-3,17-dione and 4-acetoxy-4-androstene-3,17-dione and sustained effects in vivo

4-Hydroxy-4-androstene-3,17-dione (4-OHA) and 4-acetoxy-4-androstene-3,17-dione (4-AcA), in addition to being competitive inhibitors of aromatase, cause time-dependent, irreversible, loss of enzyme activity in both human placental and rat ovarian microsomes. $\text{In vivo}$, treatment of rats with 4-OHA also causes loss of ovarian aromatase activity. To test whether this loss of activity could have $\text{in vivo}$ significance, rats with hormone-dependent, mammary tumors were treated with 4-OHA on alternate weeks. Tumor regression continued to occur during the weeks without treatment. These findings suggest that inactivation of aromatase is important in the mechanism of action of the compounds $\text{in vivo}$.     

Prostaglandin synthesis in rat embryo tissue: The effect of non-steroidal anti-inflammatory drug in vivo and ex vivo

Aspirin and salicylate are well-known but poorly understood teratogens in laboratory animals. Because aspirin inhibits PG synthesis, we systematically examined PG synthesis in rat embryo homogenates, the inhibition of PG synthesis $\text{in vivo}$ and $\text{ex vivo}$ by various non-steroidal anti-inflammatory drugs, and tested the hypothesis that the inhibition of PG synthesis is responsible for aspirin-induced limb defects in rats. We report that embryonic rat homogenates synthesis 6-keto-PGF_1α, PGE, and PGF in large amounts from endogenous substrate, that aspirin and other non-steroidal anti-inflammatory drugs inhibit PG synthesis $\text{in vitro}$ but not necessarily $\text{in vivo}$, and that contrary to our original hypothesis, the inhibition of PG synthesis is likely not responsible for aspirin-induced limb defects in rats.     

Hamster oocyte maturation in vitro: Inhibition by follicular components

When prevulatory hamster follicles were cultured $\text{in vitro}$, the oocytes within them remained at the germinal vesicle stage. This maturation arrest was partly overcome by washing the follicles before cultivation or by the addition of LH to the medium. LH-reversible oocyte arrest was also induced in isolated oocytes by culturing them either with the cumulus oophorus or with hamster follicular fluid was not species-specific, inhibitory effect of follicular fluid was not species-specific, since an LH-reversible inhibition was also produced by follicular fluid of bovine origin. Evidence is presented indicating that the inhibition is due to a heat labile peptide with a molecular weight between 10 0 and 10,000. LH may induce oocyte maturation by acting on the oocyte so that it no longer responds to the inhibitor.     

Conjugation of androgens and estrogens by human breast tumors in vitro

The metabolism of ³H-androstenedione (Δ⁴ -A) and ³H-estriol (E³) was studied in 12 human breast tumors. Part of each tumor was analyzed for estrogen receptor content. Aliquots of tumor homogenates were incubated for 2 hr separately with ³H-δ⁴-A and ³H-E₃ in the presence of appropriate cofactors. No distinct differences emerged in the profiles of the unconjugated metabolites of ³H-δ⁴-A, the major compounds in the approximate order of descendence being androsterone, androstanedione, testosterone, 5α-androstane-3α,17β-diol, epiandrosterone, and dihydrotestosterone. One tumor homogenate from an infiltrating lobular carcinoma converted ³H-Δ⁴-A to glucosiduronate metabolites (11%), of which androsterone, 6.4%; testosterone, 1.6%; and androstanediol, 0.6% predominated. The homogenate of this tumor and two other tumors converted ³H-E₃ to ³H-E₃-3S. Conversions of E₃ to E₃-3S In the other tumor homogenates were less than 0.6%. No correlation between receptor content and the capability of the tumor to conjugate Δ⁴-A or E₃ evolved. However, correlations between steroid hormone metabolism and tumor histopathology may exist.     

N-hydroxymethylpentamethylmelamine,a major in vitro metabolite of hexamethylmelamine

N-Hydroxymethylpentamethylmelamine (HMPMM) was identified by HPLC and by GLC-MS after derivatization, as a metabolite of the anticancer drug hexamethylmelamine (HMM) in incubation mixtures with fortified mouse liver 9000 × g and microsomal preparations. HMPMM formation was dependent on the presence of NADPH and oxygen. N-demethylated metabolites were also found. HMPMM displays appreciable chemical stability and 29% was recovered after 60 min incubation in buffer. HMPMM constituted more than 50% of total HMM metabolites in 30 min incubations. The known chemical reactivity of carbinolamines means that HMPMM could be involved in the pharmacological or toxic effects of HMM.